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Isolation and characterization of NBS-LRR resistance gene analogs from sugarcane.pdf

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Isolation and characterization of NBS-LRR resistance gene analogs from sugarcane

RESEARCH PAPER ACTA AGRONOMICA SINICA Volume 35, Issue 4, April 2009 Online English edition of the Chinese language journal Cite this article as: Acta Agron Sin, 2009, 35(4): 631–639. Received: 28 August 2008; Accepted: 13 December 2008. * Corresponding author. E-mail: xlpmail@ Copyright ? 2009, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at /zwxb/ DOI: 10.1016/S1875-2780(08)60076-0 Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane QUE You-Xiong, XU Li-Ping*, LIN Jian-Wei, and CHEN Ru-Kai Key Laboratory of Sugarcane Genetic Improvement, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China Abstract: For the purpose of isolating resistance gene analogs (RGAs) from sugarcane (Saccharum officinarum Roxb.) with primers from the conservative sequences of nucleotide-binding site (NBS), 6 forward and 10 backward primers were designed according to the conserved motifs in the NBS regions of 3 typical NBS-LRR resistance genes RPS2, N, and L6. The homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA of smut-resistant sugarcane variety NCo376. A total of 11 RGAs were obtained, of which 5 from the genomic DNA and 6 from the cDNA. Sequence analysis showed that these RGAs comprised of the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type of disease-resistance gene. The last amino acid in the alignment was residue W in LLVLDDV (W/D) motif, which is typical in non-TIR-NBS-LRR type of genes, indicating only non-TIR-NBS-LRR type resistance genes in the genome of sugarcane. The 11 RGAs, together with RPS2 and Xa1, were clustered into one group, and N and L6 were in another group. One RGA, termed PIC (EF059974), was validated through real-time PCR. The result showed that the expression of PI